Biology: Chapter 3: Enzymes: Immobilizing enzymes
- Immobilized enzyme: Enzymes attached to an inert, insoluble molecule so they do not get mixed in the solution of substrates and product, which is cost-effective because enzymes are not wasted.
- Using immobilized enzymes means you can re-use the enzymes while the product is enzyme free.
Lactase
- Lactase is mixed with sodium alginate.
- Droplets of this mixture is added to a solution of calcium chloride.
- Each droplet instantly reacts with the calcium chloride to form a jelly bead, containing the enzyme.
- These beads can be packed into a column, where milk, containing Lactases' substrate, lactose, is poured over them.
- Lactase in the alginate beads convert the lactose into glucose and galactose, and trickles down the column, where it can be collected as lactose-free milk.
- This is necessary for producing lactose-free dairy products for people who are lactose-intolerant. Without immobilizing the lactase, the milk would be contaminated with lactase, which we also will be unable to re-use.
- Another advantage of immobilization is that immobilized enzymes are more tolerant of different pH and temperature changes, because their molecules are held more firmly in place by the alginate they are embedded in and less exposed to the external environment, which means they are less likely to denature.
Biology: Chapter 3: Enzymes: Inhibitors
- Enzyme inhibitors reduce the rate of reaction by interfering with the enzyme in some way.
- Can be temporary or permanent.
Competitive inhibitors
- Another molecule, with a similar structure to the type of substrate the enzyme catalyses, binds with the enzyme, inhibiting it's function for other substrates.
- The competitive inhibitor therefore creates competition for the substrate, lowering the rate of reaction as the substrates have less enzymes to bind to.
- Rate of reaction depends on the concentration of substrate and inhibitor. More inhibitors - lower rate of reaction.
- Usually temporary as the inhibitor will leave the enzyme after a certain time.
- Example: Ethylene glycol is used as antifreeze, and can sometimes be drunk accidentally. In the body it is quickly converted to oxalic acid, which causes permanent kidney damage. However, when the person is given ethanol, ethanol acts as a competitive inhibitor, slowing down the reaction of ethylene glycol long enough for it to be excreted.
Non-competitive inhibitors
- Molecule that binds to another part of the enzyme, called the Allosteric site, affecting the bonding in the 3D shape of the enzyme molecule and altering the shape and shape of the active site so substrates cannot bind to the enzyme.
- Enzyme will be blocked no matter how much substrate is present, which is why it is called a non-competitive inhibitor.
- Usually permanent, denaturing the enzyme they exhibit, which can be lethal.
- However, temporary non-competitive inhibitors are essential for metabolic reactions. This is because reactions need to tightly controlled as so they don't 'run wild'.
- Temporary non-competitive inhibitors allow products to be produced in specific amounts.
- In multiple sclerosis, enzyme rate is not controlled and the immune system allows the enzymes to attack the nerves, resulting in paralysis.
- One common way of controlling metabolic reaction is using the end product of a metabolic pathway as a non-competitive inhibitor.
- Metabolic pathway: Process composed of many different reactions, each catalysed by a different enzyme.
- This means the process controls itself, as the more product is produced, the more enzymes get inhibited, meaning less substrate binding. But once the end-product is needed, it can detach from the enzyme, allowing it to function again.
Biology: Chapter 3: Enzymes: Factors that affect enzyme activity
Course of a reaction
- In the beginning of the reaction, there are a large number of substrate molecules, so every enzyme has a substrate molecule to bind with, so rate of reaction is fastest at the beginning.
- But as the reaction continues, the substrate level goes down because they are being converted to products, so there are fewer substrates to bind to enzymes, reaction rate decreases, until it completely stops when all substrates have been converted to product.
- Initial rate of reaction: The rate at the beginning of the reaction, because rate is always quickest at the beginning. This can be found by drawing a tangent at the curve as close to time 0 as possible and calculating the gradient.
Enzyme concentration
- Rate of reaction will increase with a higher enzyme concentration if there is always an excess of substrate concentration.
- Initial rate of reaction increases.
- If there is an limiting factor, eg. substrate concentration, the rate will not exceed after a certain point. For substrate concentration this is because even if you increase enzyme concentration, there will only be a limited amount of substrates to bind to the enzymes, and after the point where each enzyme has a substrate to bind to, excess enzymes will be useless because each substrate already has an enzyme to bind to.
- Rate of reaction slowly decreases as substrate gets converted to product and in the end the total amount of product will be the same because the same amount of substrate was used.
- Effect of enzyme concentration can only be fairly measured at the beginning, because initial rate of reaction will vary the most with different enzyme concentrations.
Substrate concentration
- Rate of reaction will increase with a higher substrate concentration if there is an excess of enzyme concentration.
- Initial rate of reaction increases.
- There are more substrates for enzymes to bind to, limited to amount of enzymes for substrates to bind to.
- If more substrates are added than the amount of enzyme, each enzyme will have a substrate. The enzyme cannot work any faster, and substrates will need to wait for the enzyme to finish catalysing the first substrate.
- Enzyme will be working at it's fastest possible rate, known as Vmax.
Temperature
- Reaction is slower at low temperature: Less kinetic energy means molecules move slowly, so there are less collisions between enzymes' active site and substrates.
- Reaction speeds up as temperature increases: There is more kinetic energy, so molecules move faster therefore there are more frequent collisions.
- However, at a certain temperature, enzyme will denature and stop working: Above a certain temperature, there is a large amount of kinetic energy, which also means a large amount of Vibrational energy. The structure of the enzyme molecule vibrates so much that some of the bonds holding the precise 3D shape of the enzyme molecule, especially the hydrogen and ionic bonds, start to break, This changes the shape of the enzyme, and therefore the shape of the active site, and substrates will no longer be able to fit into the active site. This is called denaturing, and is often irreversible.
- Optimum temperature: Temperature that the enzyme works best and fastest in. Often the temperature before the enzyme starts denaturing.
- In the human body, the optimum temperature of enzymes is our body temperature (37C), however, enzymes in different conditions will have different optimum temperatures.
pH level
- pH level: Amount of hydrogen ions in the solution. The lower the pH, the higher the hydrogen ion concentration (H+). The higher the pH, the higher the Hydroxide ion concentration (OH-). Measure of acidity and basicity.
- Hydrogen and hydroxide ions can interact with the R groups of amino acids by affecting the ionisation (charges) of the group.
- This affects the ionic bonding between groups, which can alter the 3D structure of the enzyme molecule, therefore also altering the shape of the active site.
- Small differences in pH are reversible because the bonds can be reformed, However, a pH that is very different from the optimum pH of the enzyme can denature the enzyme.
- Optimum pH value: When the hydroxide and hydrogen ions positively affect the bonds in the enzyme in such a way they make the active site more suited to the shape of the substrate.
Biology: Chapter 3: Enzymes: Basics
- Enzyme: Protein molecules that act as biological catalysts.
- Biological catalyst: Molecule which speeds up a chemical reactions and remains unchanged at the end of the reaction.
Features of enzymes
- Intracellular: Enzymes that operate within cells.
- Extracellular: Enzymes secreted by cells and catalyse reactions outside cells.
- Globular: Hydrophilic R groups on the outside - Soluble
- Active site: Region (cleft or depression) that another molecule can bind to.
- Substrate: Molecule that binds to enzyme
- Enzyme-substrate complex: Binding of substrate and enzyme through temporary bonding between the R groups of the enzyme's amino acids and the substrate.
- Product: What you're left with after substrate binds with enzyme - Product of the reaction
- Anabolic: Making bonds
- Catabolic: Breaking bonds
Induced fit
- Lock and key hypothesis: Each type of enzyme is specific to only one type of substrate; only one type of substrate fits in each type of enzyme's active site. However, this has been shown to be not exactly accurate, but instead enzymes and substrates have an induced fit.
- Induced fit: Enzyme, and sometimes substrate, can change shape slightly as the substrate enters the active site to ensure a perfect fit.
- Activation energy: Energy needed before a reaction occurs
- Many necessary substrates in the body need a higher temperature than 37C before they can react, but it is impossible for us to raise our body temperature that high.
- Enzymes lower the activation energy without increasing the heat by holding the substrates or substrate in such a way (physically breaking the bonds) they catalyse or by adding another chemical (H20 for hydrolysis/ ions).
Examples of enzymes
- Lactase: Breaks lactose into glucose and galactose. Found in the small intestine.
- Catalase: Breaks Hydrogen Peroxide into water and oxygen. Found in all living organisms.
